Integrated liquid mixing supply module for in-situ liquid phase transmission electron microscopy

Abstract text (incl. figure legends and references)

Advances in in-situ LPEM have made it possible to image a wide variety of dynamic processes in liquid samples. One dynamic parameter of interest is the chemical composition of the liquid surrounding the sample. Liquid composition can be controlled through flow or by mixing liquids prior to the imaging area. Current mixing strategies in LPEM rely on the use of syringe pumps and sample carriers with "bathtub" designs. They otherwise rely on pre-mixing on a sample vial or on combining two adjacent cells with small amounts of liquid under the electron beam. However, certain processes such as chemical precipitation induce changes shortly after combining the reagents and require constant refreshing of the liquid in the sample area. To overcome this limitation, we present the Liquid Supply System (LSS) with integrated liquid mixing. This technology allows to change the composition of the liquid environment on-the-fly and improves the controllability of liquid properties. Also, thanks to the design of the Nano-Cell sample carrier, it is guaranteed that the mixed phase passes through the imaging area. The LSS with liquid mixing consists of two individual liquid lines, each carrying one reagent. The liquid lines are combined in a mixing element prior to reaching the window area. Each liquid line is equipped with a precise pressure-based microfluidic flow control system that allows fine control of the concentration of the resulting mixture. We show the advantages of the liquid mixing module applied to nucleation and growth of calcium carbonate, a phenomenon of great interest across several academic and industrial fields. For this study, we use the Stream system, which consists of a smart MEMS-based liquid sample carrier, a Stream holder and the LSS with integrated liquid mixing. To study the early stages of calcium carbonate nucleation and growth we use two sample vials. One is filled with 50 mM of sodium bicarbonate (NaHCO3), dissolved in Milli-Q water, and the other with 50 mM of calcium chloride anhydrous (CaCl2), also dissolved in Milli-Q water. We use a Thermo Fisher Scientific Tecnai T20 G2, 160 kV TEM for image acquisition. We first flow a 1:1 mixture of both reagents at a rate of 10µl/min which induces the growth of calcite. We then replace the liquid with air, change the ratio of the mixture to 1:2 (double the amount of calcium chloride) and observe the formation of amorphous calcium carbonate as well as a lower number of calcite crystals. Our results show unprecedented control over the concentration of the mixed phase and allows to control kinetics-on-the-fly. The design of the Stream system guarantees that a predictable flow path is followed increasing the reproducibility of experiments. We expect that this technique will have applications not only in the field of biomineralization but in the fields of nanoparticle nucleation, growth, self-assembly and coarsening, in Liquid–Liquid Phase Separation of proteins, peptide self-assembly, vesicles studies and others.

Figure 1 legend: Calcite particles resulting from a 1:1 mixture of sodium bicarbonate and calcium chloride (top) with diffraction (bottom)

Figure 2 legend: Amorphous calcium carbonate (top) and calcite (bottom) particles obtained from flowing a 1:2 mixture of sodium bicarbonate and calcium chloride. Corresponding Diffraction in inset